Particulate supports used in separation processes, especially those involving biological materials, are advantageously characterized by the properties of being derivatizable to desired affinity ligands and ability to permit the free flow of fluids which may contain macromolecules and/or particles that desirably are not retarded mechanically by the support. Thus, the solid support should be capable of providing surface functional groups which can be used to link the support to the desired ligand. The support should also have a sufficient surface area to provide a high concentration of said ligands, but should provide passages for fluids which do not either retard contaminants or, worse, preclude access of the substance desired to be absorbed to the affinity ligand to the position of the ligand on the support. Thus, if the surface area is achieved by supplying particles containing pores, the pores must be of sufficient size to accommodate the contents of the fluid to be treated.
Because the above characteristics are most easily achieved by using rigid spherical particles of adequate size to provide reasonable flow rates, adequate surface area is best achieved by insuring that these particles are porous. The surface of the pores may then be employed to accommodate the affinity ligands. Preparation of such porous, rigid, and spherical solid supports have utilized several approaches.
In one approach, polymerization is conducted in a suspension, but in the presence of one or more agents designed to generate pores in the polymerized, finished product. This approach is described in U.S. Pat. Nos. 4,224,415; 4,256,840; 4,297,220; and 4,382,124. Soluble polymers have been used to function as porogens as described in U.S. Pat. No. 4,104,209. Reverse micellular systems obtained by adding water and suitable surfactant to a polymerizable monomer have been described as porogens by Menger et al., J Am Chem Soc (1990) 112:1263-1264.
Alternative approaches have used thermal induction of phase separation to generate porous substances as described in European Patent Application 370,259 or U.S. Pat. No. 4,430,451.
More closely related to the approach of the present invention is the application of template polymerization. This procedure, described, for example, in U.S. Pat. No. 4,263,268 and U.S. Pat. No. 4,933,372, relies on the use of an inorganic porous particle as a template, in the pores of which the desired organic polymer is polymerized. When the inorganic template is removed (by dissolution in an appropriate solvent) the remaining organic particles are formed in the image of the pore structure of the inorganic support. The pores resulting in these particles are relatively small as they relate to the dimensions of the nonporous substance of the template particles and the size of the individual particles is governed by the size of the individual inorganic templates.
The method of the present invention overcomes these difficulties by providing a means to obtain particles of larger dimensions and having pores of greater diameter, nevertheless networked throughout the particle. Disclosure of the Invention
The invention provides large, rigid macroporous polymer beads wherein the pore size is generally larger than one micron in diameter. The results beads are of sufficient size and of sufficient pore size to permit ready flow of liquids through them, and to accommodate high densities of affinity ligands coupled to the surface external to the particles and internal to the pores. These macroscopic particles are obtained by first filling the pores of inorganic porous particles with a "blowing agent"--a temporary filler which will be moved at the end of the preparation. The inorganic particles, with pores blocked, are then used per se to create the internal pores in a macroparticle formed around a collection of such inorganic particles. After polymerization of the organic component to obtain the macroparticles, the blowing agent is activated to liberate the pores and to disrupt that portion of the polymerized materials which separates the inorganic particles. Thus, channels are created which connect the inorganic particles and the high-diameter pores are then formed throughout the polymer bead as the template particles are removed.
Thus, in one aspect, the invention is directed to a composition which comprises macroscopic beads of crosslinked organic polymer which are permeated by megapores. These particles may further be derivatized to contain, at the surfaces of the pore and of the exterior, functional groups which permit covalent binding of coupling agents or affinity ligands to the surfaces. The particles may further contain ligands thus coupled. These beads are useful in a variety of applications wherein separation, analysis, alterations of concentration, and the like are desired.
Thus, in other aspects, the invention relates to methods to conduct separation or analysis using the beads of the invention. The invention further includes devices or containers configured so as to accommodate the invention beads and containing these beads. In still another aspect, the invention is directed to methods to prepare the macroscopic beads.